Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: High expression levels of RTKs and NRF2 correlate with worst prognosis in TNBC patients. A Volcano plot for Spearman correlation between NRF2 expression and indicated RTKs in BC samples from TCGA-BRCA dataset. Significant positive correlations are shown in red, and negative correlations in blue. Common RTKs are highlighted. B Expression levels of MET and EGFR across the four different BC subtypes from the TCGA dataset. Kaplan–Meier curves showing the probability of overall survival in TNBC patients with different expression levels of MET/NRF2 (left) and EGFR/NRF2 (right) ( C ), AXL , TGFBR1 , PDGFRA , and NRF2 ( D ). Statistical analysis: ( B ) One-way ANOVA followed by Tukey’s multiple comparison statistical test was performed. C-D Survival data derived from GSE31519 dataset for TNBC patients. P values were computed using the Logrank (Mantel Cox) test. ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Expressing, Comparison, Derivative Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: Inhibition of the MET receptor reduces NRF2 nuclear localization and activity in TNBC MMTV-R26 Met cell lines. A Immunoblotting (left) of pY 1234/35 MET, MET, NRF2, and relative densitometric analysis (right) of NRF2 protein levels in all MGT cell lines derived from the MMTV-R26 Met tumours. Actin was used as a loading control. B Immunoblotting (left) of pY 1234/35 MET, MET, NRF2, and relative densitometric (right) analysis of NRF2 protein levels in the MGT-13 cell line upon 16 h of PHA treatment. Actin was used as a loading control. C Immunofluorescence (left) and relative quantification (right) of NRF2 (red) nuclear intensity in MGT-13 cells upon 16 h of PHA treatment. DNA (Hoechst, blue). D Immunoblotting (top) and relative densitometric analysis (bottom) of NRF2 cytosolic and nuclear fractions in MGT-13 cells upon 16 h of PHA treatment. Vinculin and Lamin A/C were used as loading and quality controls. ( E ) RT-qPCR of NRF2 target genes in MGT-13 cells after 16 h of PHA treatment. Actin was used as housekeeping gene. F Workflow of RNA-seq experiments. p-value ≤ 0.5 and Log 2 foldchange (log₂FC) > 0.7 were used as threshold for analysis. G Activation z-score for transcription factors (CTRL vs. PHA). Values lower than − 2 (blue bars) suggest an inhibition of the pathway related to the corresponding transcription factor in the PHA treatment. Values over 2 (red bars) suggest an activation of the pathway in PHA samples. NFE2L2 (NRF2) is indicated in bold. Data from IPA. H Heatmap of z-scores for genes involved in the NRF2 pathway (according to IPA) for CTRL vs. PHA (3 samples for each condition, see x axis). Positive values of z-score (red) indicate upregulation within the sample, negative values (blue) downregulation. Results represent the mean of at least three independent experiments (± SEM or ± SD). Statistical analysis: ( A-B-D ) Unpaired t -test. C Mann-Whitney test according to the normal distribution. E Multiple t- test. PHA: PHA-665752 1 µM, MET inhibitor. ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Inhibition, Activity Assay, Western Blot, Derivative Assay, Control, Immunofluorescence, Quantitative Proteomics, Quantitative RT-PCR, RNA Sequencing, Activation Assay, MANN-WHITNEY

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: Targeting of the MET receptor reduces NRF2 activity in human TNBC cell lines. A Immunoblotting analysis of pY 1234/35 MET, MET, and NRF2 in human BC cell lines. Ponceau was used as a loading control. B Immunofluorescence (left) and relative quantification analysis (right) of NRF2 (red) nuclear intensity in human TNBC cells upon 16 h of PHA treatment. DNA (Hoechst, blue). C RT-qPCR of NRF2 target genes in human TNBC cells after 16 h of PHA treatment. Actin was used as housekeeping. D Immunoblotting (top) analysis of pY 1234/35 MET, MET, and NRF2 and relative densitometric analysis (bottom) of NRF2 in human BC cell lines after 4 h of serum-free media and 10 min of HGF stimulation (50ng/mL). Vinculin was used as a loading control. E Clonogenic assays (top) and relative quantification of the number of colonies (bottom) on MDA-MB-231, BT-549 and MCF10-A cells treated with PHA and ML-385 for 72 h. Results represent the mean of at least three independent experiments (± SEM or ± SD). Statistical analysis: ( B ) Mann-Whitney test according to the normal distribution. C Multiple t -test. D Unpaired t -test. E One-way ANOVA statistical test. PHA: PHA-665752 1 or 2 µM, MET inhibitor. ML-385: 5 µM, NRF2 inhibitor. ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Activity Assay, Western Blot, Control, Immunofluorescence, Quantitative Proteomics, Quantitative RT-PCR, MANN-WHITNEY

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: MET or NRF2 targeting enhances Paclitaxel sensitivity of TNBC cells. Cell viability of MGT-13 ( A ) and BT-549 ( B ) cells exposed to PTX in combination with PHA or ML-385. Percentage of cell viability in presence of drugs compared to controls (untreated cells) is indicated using a colour scale (from green to red). C-D Top panel: matrix synergy plot representing the synergy/antagonism score of each combination and its statistical significance calculated by Loewe model. Bottom panel: mapped to d-r surface showing the synergy distribution of drug combinations. The synergistic analysis was performed on MGT-13 ( C ) and BT-549 ( D ) cell lines. E-F Clonogenic assays on MGT-13 cells exposed to PTX in combination with PHA ( E ) or ML-385 ( F ) and treated as in Fig. 4A. G-H Histograms of flow cytometry experiments representing the percentage of dead cells upon ANNEXIN V/DAPI + staining of MGT-13 cells exposed for 72 h to PHA and PTX ( G ) or ML-385 and PTX ( H ) treatments alone or in combination. Results represent the mean of at least three independent experiments (± SEM). Statistical analysis: ( A-B-G-H ) One-way ANOVA statistical test was performed for each combination compared to the drug alone. (In A-B, * indicate the significance respect to PTX treatment). C-D Loewe models were used for synergy score calculation by Combenefit software. PHA: PHA-665752: MET inhibitor; ML-385: NRF2 inhibitor. PTX: Paclitaxel. ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Staining, Software

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: MET-NRF2 axis inhibition increases ROS levels and DNA damage accumulation. Immunoblotting ( A ) and relative densitometric analysis ( B ) of p62 and HO-1 in MGT-13 and BT-549 cells exposed for 24 h to PTX in combination with PHA. Actin was used as a loading control. C-F Cytofluorimetric analyses (top) and relative histograms (bottom) of reactive oxygen species (ROS) upon DHE staining on MGT-13 ( C-D ) and BT-549 ( E-F ) cells exposed for 24 h to PTX in combination with PHA or ML-385. Confocal microscopy analysis ( G ) and relative quantification ( H ) of 8-oxo-DG (red) foci/cell in MGT-13 cells treated alone or in combination with PTX and PHA. DNA (Hoechst, blue). Confocal microscopy analysis ( I ) and relative quantification ( J ) of γH2AX (green) positive cells in MGT-13 cells treated alone or in combination with PTX and PHA. K Immunoblotting analysis of γH2AX in MGT-13 and BT-549 treated as in ( I ). Vinculin and GAPDH were used as loading control. Results represent the mean of at least three independent experiments (± SEM or ± SD). Statistical analysis: ( B-D-F-H-J ) One-way ANOVA statistical test was performed for each combination compared to the drug alone. PHA: PHA-665752 1 or 3 µM, MET inhibitor. ML-385: 5 µM, NRF2 inhibitor. PTX: Paclitaxel 3nM or 30nM. ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Inhibition, Western Blot, Control, Staining, Confocal Microscopy, Quantitative Proteomics

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: MET modulates NRF2 signalling via SRC kinase-dependent p62/KEAP1 pathway. Immunoblotting ( A ) of pY 1234/35 MET, MET, pY 416 SRC, SRC and relative densitometric analyses ( B ) of pY 416 SRC normalized on total SRC in TNBC cell lines after 16 h of PHA treatment. GAPDH was used as loading control. C Immunoblotting pY 1234/35 MET, MET, pY 416 SRC, SRC, and NRF2 in MGT-13 cells transiently transfected with empty vector (pSGT) and active SRC (SRC Y527F ) after 16 h of PHA treatment. Actin was used as loading control. Immunoblotting ( D ) and relative densitometric analyses ( E ) of KEAP1, pS 349 p62 and p62 in human TNBC cell lines after 16 h of PHA treatment. Actin was used as loading control. F Confocal microscopy analyses and relative quantification of colocalizing dots of KEAP1 (red) and p62 (green) in human TNBC cell lines upon 16 h of PHA treatment. DNA (Hoechst, blue). 3X digital magnification showing merged signals. Results represent the mean of at least three independent experiments (± SEM or ± SD). Statistical analysis: ( B-E ) Unpaired t -test. F Mann-Whitney test according to the normal distribution. PHA: PHA-665752 1 µM, MET inhibitor. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Western Blot, Control, Transfection, Plasmid Preparation, Confocal Microscopy, Quantitative Proteomics, MANN-WHITNEY

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: SRC expression sustains NRF2 nuclear accumulation and activity in TNBC. A Immunoblotting (top) of NRF2, pY 416 SRC, SRC and relative densitometric analysis (bottom) of NRF2 protein levels in MDA-MB-231 cells upon 16 h of DAS treatment. GAPDH was used as a loading control. B Immunofluorescence (left) and relative quantification (right) of NRF2 (red) nuclear intensity in MDA-MB-231 cells upon 16 h of DAS treatment. DNA (Hoechst, blue). C Immunoblotting (top) and relative densitometric analysis (bottom) of NRF2 cytosolic and nuclear fractions in MDA-MB-231 cells upon 16 h of DAS treatment. Vinculin and Lamin A/C were used as loading and quality controls. D RT-qPCR of NRF2 target genes in MDA-MB-231 cells after 16 h of DAS treatment. Actin was used as housekeeping gene. E Expression levels of SRC in log 2 across the four different breast cancer subtypes from the TCGA dataset. F-G Kaplan–Meier curves show the overall survival probability of TNBC ( n = 579; F ) and non-TNBC ( n = 544; G ) patients with different expression levels of SRC and NRF2 . Results represent the mean of at least three independent experiments (± SEM or ± SD). Statistical analysis: ( A-C ) Unpaired t- test. B Mann-Whitney test according to the normal distribution. D Multiple t -test. E One-way ANOVA followed by Tukey’s multiple comparison statistical test was performed. F-G Survival data obtained from Gene Expression Omninbus (GEO) Id GSE31519 for TNBC patients and TCGA dataset for non-TNBC patients. P values were computed using the Logrank (Mantel Cox). DAS: Dasatinib 50 nM; SRC inhibitor. ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Expressing, Activity Assay, Western Blot, Control, Immunofluorescence, Quantitative Proteomics, Quantitative RT-PCR, MANN-WHITNEY, Comparison, Gene Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: MET, SRC or NRF2 targeting improves Paclitaxel efficacy in TNBC patient-derived organoids. Organoid viability assays of PDO-21, PDO-43, and PDO-46 after PHA and PTX ( A ), ML-385 and PTX ( B ), DAS and PTX ( C ) combination treatments. D 4X digital magnification of bright-field images and relative quantification of organoids size of PDO-21 culture treated as described in ( A-B-C ). Scale bar: 400 μm. E Percentage of small, medium, and large PDO-21 treated as in ( A - B - C ). F Working model. Results represent the mean of at least three independent experiments (± SEM). Statistical analysis: ( A-B-C ) One-way ANOVA statistical test was performed for each combination compared to the drug alone. E Two-way ANOVA followed by Tukey’s multiple comparison test (* respect to CTR, $ respect to inhibitors and # respect to PTX). PHA: PHA-665752, MET inhibitor. DAS: Dasatinib. ML-385: NRF2 inhibitor. PTX: Paclitaxel. ns: not significant; *, $, # p < 0.05; **, $$, ## p < 0.01; ***, $$$, ### p < 0.001; ****, $$$$, #### p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Quantitative Proteomics, Comparison

Journal: Cell metabolism

Article Title: The Mitochondrial-Encoded Peptide MOTS-c Translocates to the Nucleus to Regulate Nuclear Gene Expression in Response to Metabolic Stress

doi: 10.1016/j.cmet.2018.06.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: NFE2L2 Human siRNA Oligo Duplex , Origene , Cat#SR321100.

Techniques: Recombinant, SYBR Green Assay, Reporter Assay, Over Expression, Sequencing, Luciferase, Software, Real-time Polymerase Chain Reaction

Journal: Cell metabolism

Article Title: The Mitochondrial-Encoded Peptide MOTS-c Translocates to the Nucleus to Regulate Nuclear Gene Expression in Response to Metabolic Stress

doi: 10.1016/j.cmet.2018.06.008

Figure Lengend Snippet:

Article Snippet: NFE2L2 Human siRNA Oligo Duplex , Origene , Cat#SR321100.

Techniques:

Journal: Nature Communications

Article Title: PPIA dictates NRF2 stability to promote lung cancer progression

doi: 10.1038/s41467-024-48364-4

Figure Lengend Snippet: A Representative immunoblot analysis of NRF2 protein levels in nuclear extracts of 17 NSCLC cell lines. B Dot plot showing the relative nuclear NRF2 protein levels in cell lines with KEAP1 and/or KRAS co-mutations ( n = 9 cell lines) and in cell lines with KEAP1 and KRAS WT ( n = 8 cell lines) related to Fig. 1A. C Overview of the Ds R ed- I RES-E G FP-NRF2 (RIG-NRF2) screen. CMV, CMV promoter; IRES, Internal ribosome entry site. D Flow cytometry analysis of DsRed and EGFP levels of A549-RIG-NRF2 cells treated with CHX (10 μM), MG132 (10 μM), Ki696 (1 μM), ML334 (50 μM) and CsA (10 μM). The gating strategy are provided in Supplementary Fig. . E IC 50 values of CsA against a panel of NSCLC cell lines with distinct genetic profile. F Representative immunoblot analysis of NRF2 protein levels in a panel of NSCLC cells upon CsA treatment (10 μM). The results of panels ( A , D and F ) are representative of three independent experiments. E represents mean ± SD of three independent experiments. P value was analyzed using Two-tailed unpaired Student’s t-test, P < 0.05 was considered statistically significant. Source data are provided as a Source Data file.

Article Snippet: siRNA negative control and siRNA targeting NRF2, PPIA, KLF5 were obtained from Shanghai Synbio Technologies.

Techniques: Western Blot, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: PPIA dictates NRF2 stability to promote lung cancer progression

doi: 10.1038/s41467-024-48364-4

Figure Lengend Snippet: A Representative immunoblot analysis of NRF2 protein levels in PPIA- WT or PPIA -KO A549 cells. Quantitative data was provided in the right panel. B Representative immunoblot analysis of NRF2 protein levels in PPIA- WT or PPIA KO A549 cells treated with CsA (0, 3, 10 μM) for 48 h. Quantitative data was provided in the right panel. C Immunoprecipitation of NRF2 followed by immunoblot analysis with anti-ubiquitin antibody detected the NRF2 ubiquitination in PPIA -WT and PPIA -KO A549 cells. D CHX chase assay of NRF2 protein stability in PPIA -WT or PPIA -KO A549 cells. Cells were treated with CHX (100 μg/mL) at the indicated time points and then subjected to immunoblot. Quantitative data was provided in the right panel. E Representative immunoblot results demonstrate that PPIA WT, but not PPIA catalytically dead PPIA variant (PPIA R55A&F60A ), can restore the NRF2 level in PPIA -KO A549 cells. Quantitative data was provided in the right panel. F Schematic diagram of NanoBRET™ Ubiquitination Assay using the HaloTag®-Ubiquitin and NRF2-NanoLuc® fusion construct. G BRET signal of HEK293T cells transfected with HaloTag®-Ubiquitin and NRF2-Nanoluc® following si PPIA treatment or not. H Immunoprecipitation of NRF2 followed by immunoblot analysis with anti-ubiquitin antibody detected the NRF2 ubiquitination in HEK293T following si PPIA treatment or not. I Relative cell growth of A549 upon PPIA KO or CsA (10 μM) treatment in the presence or absence of NRF2 overexpression. Quantitative results were shown in upper panel and representative colony image was presented in lower panel. C and H are representative of three independent experiments. ( A – E ), G and ( I ) represent mean ± SD of three independent experiments. P values were analyzed using Two-tailed unpaired Student’s t-test, P < 0.05 was considered statistically significant. Source data are provided as a Source Data file.

Article Snippet: siRNA negative control and siRNA targeting NRF2, PPIA, KLF5 were obtained from Shanghai Synbio Technologies.

Techniques: Western Blot, Immunoprecipitation, Ubiquitin Proteomics, Variant Assay, Construct, Transfection, Over Expression, Two Tailed Test

Journal: Nature Communications

Article Title: PPIA dictates NRF2 stability to promote lung cancer progression

doi: 10.1038/s41467-024-48364-4

Figure Lengend Snippet: A Co-immunoprecipitation analysis of endogenous proteins of PPIA and NRF2 using A549 cell lysates. B Pull-down assay of PPIA WT/NRF2 and PPIA R55A&F60A /NRF2. A549 cell lysate was incubated with PPIA WT or PPIA R55A&F60A pre-loaded beads, and NRF2 protein retained on the beads were detected by immunoblot. C The influence of CsA on the interaction between PPIA and NRF2. A549 cell lysate were incubated with PPIA pre-loaded beads in the presence of CsA (0, 3, 10 μM) and then subjected to pull-down assay. D Schematic diagram of various NRF2 truncation. E The binding of full-length and truncated NRF2 to PPIA as determined by pull-down assay. Various NRF2 truncations were overexpressed in HEK293T cells (as described in D). F CHX chase assay of NRF2 (WT or mutant P174A) protein stability. Cells were treated with CHX (100 μg/mL) at the indicated time points and then subjected to immunoblot (upper panel). Quantitative data was provided in the lower panel. G Crystal structure of PPIA in complex with NRF2 fragment PPIA-Binding Motif (PBM) ( 169 VAQVAPVD 176 ). PBM peptide is deeply embedded into catalytic pocket of PPIA presented as gray surface. 172 VAPV 175 residues of NRF2 peptide are displayed as slate stick. H Expansion of the catalytic pocket of PPIA in complex with NRF2 fragment PBM. PPIA is shown in gray cartoon and the interacted residues are displayed as salmon sticks. NRF2 PBM fragment is presented as slate sticks. The black dashed line denotes the hydrogen contact. The results of panels ( A – C , E , F ) are representative of three independent experiments. F represents mean ± SD of three independent experiments. P values were analyzed using Two-tailed unpaired Student’s t-test, P < 0.05 was considered statistically significant. Source data are provided as a Source Data file.

Article Snippet: siRNA negative control and siRNA targeting NRF2, PPIA, KLF5 were obtained from Shanghai Synbio Technologies.

Techniques: Immunoprecipitation, Pull Down Assay, Incubation, Western Blot, Binding Assay, Mutagenesis, Two Tailed Test

Journal: Nature Communications

Article Title: PPIA dictates NRF2 stability to promote lung cancer progression

doi: 10.1038/s41467-024-48364-4

Figure Lengend Snippet: A The cell viability of A549 cells upon CsA (10 μM) treatment or upon PPIA KO in the presence of glutamine (2, 1, 0.5 mM) was measured by MTT assay. Gln, glutamine. B Oxygen consumption rate (OCR) plotted over time in A549 cells following CsA (10 μM) treatment or PPIA KO. C Heatmap showing the mRNA levels of different glutamine membrane transporters in PPIA -WT or PPIA -KO A549 cells. D - E Q-PCR D and immunoblot E analysis of SLC1A5 in PPIA -WT or PPIA -KO A549 cells following NRF2 overexpression. F -G Q-PCR F and immunoblot G results of SLC1A5 in A549 cells treated with CsA (10 μM for 48 h) with or without NRF2 overexpression. H Relative glutamine levels in A549 cells upon CsA (10 μM) treatment or upon PPIA KO, following SLC1A5 or NRF2 overexpression. I A549 cells upon CsA (10 μM) treatment or upon PPIA KO following SLC1A5 overexpression were subjected to colony formation. Quantitative results were shown in upper panel and representative colony image was presented in lower panel. The results of panels ( C , E , G ) are representative of three independent experiments. A , B , D , F , H and I represent mean ± SD of three independent experiments. P values were analyzed using Two-tailed unpaired Student’s t-test, P < 0.05 was considered statistically significant. Source data are provided as a Source Data file.

Article Snippet: siRNA negative control and siRNA targeting NRF2, PPIA, KLF5 were obtained from Shanghai Synbio Technologies.

Techniques: MTT Assay, Membrane, Western Blot, Over Expression, Two Tailed Test

Journal: Nature Communications

Article Title: PPIA dictates NRF2 stability to promote lung cancer progression

doi: 10.1038/s41467-024-48364-4

Figure Lengend Snippet: A pGL3-Luc vector containing human KLF5 promoter (-4,000 to 0 bp) and pCDNA3.1-Flag-NRF2 were co-transfected into HEK293T cells with a ratio of 1:0.5 to 1:8 and luciferase activity was determined. B Analysis of NRF2 consensus motif enrichment in the KLF5 promoter (-4,000 to 0 bp) predicted by JASPAR database. Matched consensus motifs are shown in schematic. C ChIP-PCR analysis of the enrichment of NRF2 at the promoter region of KLF5 in A549 cells. D pGL3-Luc vector containing human SLC1A5 promoter (-500 to +10 bp) and pCDNA3.1-Flag-KLF5 were co-transfected into HEK293T cells with a ratio of 1:0.5 to 1:8 and luciferase activity was determined. E Analysis of KLF5 consensus motif enrichment in the SLC1A5 promoter predicted by JASPAR database. Matched consensus sequences are in bold. F ChIP-PCR analysis of the enrichment of KLF5 at the promoter region of SLC1A5 in A549 cells. G Q-PCR and immunoblot results of SLC1A5 in A549 cells treated with si NRF2 in the presence or absence of KLF5 overexpression. H Relative glutamine level in A549 cells following si NRF2 treatment in the presence or absence of KLF5 overexpression. I Colony formation of A549 cells treated with CsA or si NRF2 in the presence or absence of KLF5 overexpression. Quantitative results were shown in upper panel and representative colony image was presented in lower panel. J Correlation analysis of KLF5/NRF2 or KLF5/SLC1A5 gene expression in clinical NSCLC tumor samples ( n = 138 samples). The data are derived from public dataset (GSE8894) and analyzed in PrognoScan. The results of panels ( C , F , G ) are representative of three independent experiments. A , C , D , F , G – I represent mean ± SD of three independent experiments. P values were analyzed using Two-tailed unpaired Student’s t-test, P < 0.05 was considered statistically significant. Source data are provided as a Source Data file.

Article Snippet: siRNA negative control and siRNA targeting NRF2, PPIA, KLF5 were obtained from Shanghai Synbio Technologies.

Techniques: Plasmid Preparation, Transfection, Luciferase, Activity Assay, Western Blot, Over Expression, Gene Expression, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: PPIA dictates NRF2 stability to promote lung cancer progression

doi: 10.1038/s41467-024-48364-4

Figure Lengend Snippet: A Representative immunohistochemical analysis of PPIA and NRF2 in human LUAD tissue microarray containing lung tumor tissues and adjacent normal lung tissues. Scale bar = 50 μm. B Scatter plots showing a positive correlation of PPIA and NRF2 expression in IHC analysis of human NSCLC cancer tissues ( n = 69 samples). Linear regression with Pearson R and two-tailed P values are shown. C Kaplan-Meier survival curves of patients with NSCLC divided by high or low PPIA protein expression level according to IHC analysis ( n = 69 samples). D Kaplan-Meier survival curves of patients with NSCLC divided by high or low NRF2 protein expression level according to IHC analysis ( n = 69 samples). E – H Kaplan–Meier survival curves of patients with NSCLC based on PPIA , NRF2 , KLF5 and SLC1A5 gene expression level (for PPIA , n = 719 samples; for NRF2 , n = 672 samples; for KLF5 , n = 719 samples; for SLC1A5 , n = 719 samples). Data are integrated from Kaplan–Meier plotter ( http://kmplot.com/analysis/ ). Statistical significance for Kaplan–Meier survival curves ( C – H ) was calculated by Log-rank (Mantel-Cox) test. Source data are provided as a Source Data file.

Article Snippet: siRNA negative control and siRNA targeting NRF2, PPIA, KLF5 were obtained from Shanghai Synbio Technologies.

Techniques: Immunohistochemical staining, Microarray, Expressing, Two Tailed Test, Gene Expression

Journal: Nature Communications

Article Title: PPIA dictates NRF2 stability to promote lung cancer progression

doi: 10.1038/s41467-024-48364-4

Figure Lengend Snippet: The correlation between PPIA and NRF2 protein expression in IHC analysis of human NSCLC cancer tissues

Article Snippet: siRNA negative control and siRNA targeting NRF2, PPIA, KLF5 were obtained from Shanghai Synbio Technologies.

Techniques: Expressing

Journal: Nature Communications

Article Title: PPIA dictates NRF2 stability to promote lung cancer progression

doi: 10.1038/s41467-024-48364-4

Figure Lengend Snippet: KEAP1 , NRF2 and KRAS gene mutations among various NSCLC cell lines

Article Snippet: siRNA negative control and siRNA targeting NRF2, PPIA, KLF5 were obtained from Shanghai Synbio Technologies.

Techniques:

Journal: Nature Communications

Article Title: PPIA dictates NRF2 stability to promote lung cancer progression

doi: 10.1038/s41467-024-48364-4

Figure Lengend Snippet: X-ray data collection statistics

Article Snippet: siRNA negative control and siRNA targeting NRF2, PPIA, KLF5 were obtained from Shanghai Synbio Technologies.

Techniques:

Journal: Journal of Advanced Research

Article Title: Skin cancer biology and barriers to treatment: Recent applications of polymeric micro/nanostructures

doi: 10.1016/j.jare.2021.06.014

Figure Lengend Snippet: Visual representation of the novel biomimetic NPs based delivery system. (A) Schematic illustration of the steps involved in the preparation of biomimetic NPs utilizing various polymers and cancer cell membrane derived from the oral tongue SCC. (B) Scheme presenting the mechanisms through which the designed delivery system worked to show the combined effects of gene (Nrf2-siRNA) delivery and amplification of PDT through the delivered gene. (C1) Confocal images of the cells after 48-hour treatments with free ICG, PPI, PPI-siRNA, and M@PPI-siRNA NPs combined with laser irradiation (+L). Mitochondria stained with MitoTracker green (M7514) and Cyt c stained with anti-Cyt c antibody emitted green and red fluorescence. (C2) Quantitative analysis of released Cyt c from mitochondria in the SCC-25 cells following the treatment with NPs and laser irradiation. Red fluorescence in the merged confocal images indicated Cyt c released from mitochondria into cytoplasm. Relative released Cyt c is defined as the fluorescence intensity ratio of release Cyt c in the treated cells to that in the control cells without laser irradiation. Scale bars present 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) (D) PTT and PDT efficacies of M@PPI-siRNA NPs in SCC-25 tumor-bearing mice: (D1) IR thermal images of the mice and (D2) temperature changes inside the tumors during 10 min-laser irradiations at 8 h after IV administration of normal saline (the control), free ICG, PPI, PPI-siRNA, and M@PPI-siRNA NPs. Upon laser irradiation, M@PPI-siRNA NPs showed an improved heating efficiency as compared to other treatments, increasing the temperature up to 56.5 °C, that is sufficient for tumor ablation. (E) Tissue distributions and tumor accumulation of M@PPI-siRNA NPs in SCC-25 tumor bearing mice: (E1) Fluorescence images and (E2) mean fluorescence intensities (MFIs) of tumors and main organs collected from the mice at 24 h after various treatments. ** P < 0.01 compared to the control; # P < 0.05 for the comparison between two treatment groups. Figure is reproduced from the reference copyright © 2020 Elsevier.

Article Snippet: Considering the role of Nrf2, a redox regulated transcription factor, in the tumor resistance against PDT, Nrf2-siRNA has been chosen to design a biomimetic NPs system.

Techniques: Membrane, Derivative Assay, Amplification, Irradiation, Staining, Fluorescence, Control, Saline, Comparison

Journal: Behavioral and Brain Functions : BBF

Article Title: Forced exercise activates the NrF2 pathway in the striatum and ameliorates motor and behavioral manifestations of Parkinson's disease in rotenone-treated rats

doi: 10.1186/s12993-020-00171-9

Figure Lengend Snippet: Forward and reverse primers for real time PCR

Article Snippet: Prepared cDNA, was used in the qPCR analyzer (Step One, Applied Biosystems, Singapore) using the MAXIMA SYBR Green qPCR Master Mix with the following program: 1 cycle at 95 °C for 10 min; 40 cycles of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s; one cycle at 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s. The specific primers (Table ) of Nrf2, TFAM, NADPH dehydrogenase (NQO1 ) & housekeeping GAPDH were purchased from Metabion international AG, Germany.

Techniques:

Journal: Behavioral and Brain Functions : BBF

Article Title: Forced exercise activates the NrF2 pathway in the striatum and ameliorates motor and behavioral manifestations of Parkinson's disease in rotenone-treated rats

doi: 10.1186/s12993-020-00171-9

Figure Lengend Snippet: Expression levels of genes by PCR in the corpus striatum, effect of exercise (4 weeks treadmill), L-dopa (6 mg\kg IP daily for 4 weeks), and their combination on rotenone treated rats (2 mg\kg SC for 4 weeks). a Nrf2 gene, b expression level of NADPH dehydrogenase (NQO1) mRNA, and c expression level of TFAM mRNA, P-value is calculated by ANOVA test, and is considered significant if < 0.05. n = 10 rats in each group. *Significant when compared to control G. # significant when compared to the rotenone group. $ Significant when compared to the rotenone\exercise group

Article Snippet: Prepared cDNA, was used in the qPCR analyzer (Step One, Applied Biosystems, Singapore) using the MAXIMA SYBR Green qPCR Master Mix with the following program: 1 cycle at 95 °C for 10 min; 40 cycles of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s; one cycle at 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s. The specific primers (Table ) of Nrf2, TFAM, NADPH dehydrogenase (NQO1 ) & housekeeping GAPDH were purchased from Metabion international AG, Germany.

Techniques: Expressing, Control

Journal: Scientific Reports

Article Title: miR-155 mediates arsenic trioxide resistance by activating Nrf2 and suppressing apoptosis in lung cancer cells

doi: 10.1038/s41598-017-06061-x

Figure Lengend Snippet: Activation of the Nrf2 signaling pathway in A549 and A549R cells. The cellular location and expression of the Nrf2 signaling pathway proteins in A549 and A549R cells were detected with immunofluorescence and immunoblotting. ( A ) Nrf2 nuclear and cytoplasmic localization (400×) was detected with immunofluorescence. ( B , C ) The expression of Nrf2 in the cytoplasm and nucleus of A549 and A549R cells. The “Fold change in Nrf2 expression” was calculated based on the level of Nrf2 in A549 cells. ( D , E ) The “Fold change in protein expression” of NQO1, HO-1 in A549R cells was calculated based on the levels of the proteins in A549 cells. The blots were cropped for the clarity of the results. Results were obtained from three independent experiments, and quantitative results are presented as mean ± standard deviation (SD). “#” indicates a significant difference compared with A549 cells ( P < 0.05).

Article Snippet: Proteins were probed with primary antibodies against Nrf2, Lamin B, HO-1 and NQO1 (Boster Biological Technology, Wuhan, China, 1:500), Bcl-2, Bax (KeyGen Bio-technology, Nanjing, China, 1:200) and β-actin (ZSGB Bio, Beijing, China, 1:1000), respectively.

Techniques: Activation Assay, Expressing, Immunofluorescence, Western Blot, Standard Deviation

Journal: Scientific Reports

Article Title: miR-155 mediates arsenic trioxide resistance by activating Nrf2 and suppressing apoptosis in lung cancer cells

doi: 10.1038/s41598-017-06061-x

Figure Lengend Snippet: The effects of miR-155 on the level of Nrf2, HO-1 and NQO1 proteins in A549R cells. A549R cells were transfected with the miR-155 mimic negative control ( MC ), the miR-155 mimic ( M ), the miR-155 inhibitor negative control ( IC ) and imR-155 inhibitor ( I ). The protein levels of Nrf2, HO-1 and NQO1 were determined with immunoblotting. ( A , C ) showed representative immunoblots. ( B , D ) The “Fold change in protein expression” of Nrf2, HO-1 and NQO1, which was calculated based on the protein level in the untransfected A549R control cells. The blots were cropped for the clarity of the results. Results were obtained from three independent experiments, and quantitative results are presented as mean ± standard deviation (SD). “*” indicates a significant difference compared with the negative control groups ( MC or IC )( P < 0.05). “#” indicates a significant difference compared with untransfected A549R cells (Control) ( P < 0.05).

Article Snippet: Proteins were probed with primary antibodies against Nrf2, Lamin B, HO-1 and NQO1 (Boster Biological Technology, Wuhan, China, 1:500), Bcl-2, Bax (KeyGen Bio-technology, Nanjing, China, 1:200) and β-actin (ZSGB Bio, Beijing, China, 1:1000), respectively.

Techniques: Transfection, Negative Control, Western Blot, Expressing, Control, Standard Deviation

Journal: Scientific Reports

Article Title: miR-155 mediates arsenic trioxide resistance by activating Nrf2 and suppressing apoptosis in lung cancer cells

doi: 10.1038/s41598-017-06061-x

Figure Lengend Snippet: The pathways for miRNA-155 to mediate ATO resistance in A549R cells. miR-155 in lung cancer cells activates Nrf2 that subsequently upregulates the expression of antioxidant genes i.e., HO-1 and NQO1 in A549R cells (subpathway 1). This further increases the antioxidant capacity and promotes survival of the lung cancer cells. On the other hand, miR-155 reduces the ratio of Bax/Bcl-2 and suppressed apoptosis and cell death (subpathway 2). All these result in increased colony formation and migration, thereby leading to a high level of cellular resistance to ATO.

Article Snippet: Proteins were probed with primary antibodies against Nrf2, Lamin B, HO-1 and NQO1 (Boster Biological Technology, Wuhan, China, 1:500), Bcl-2, Bax (KeyGen Bio-technology, Nanjing, China, 1:200) and β-actin (ZSGB Bio, Beijing, China, 1:1000), respectively.

Techniques: Expressing, Migration